Therapeutic strategies to treat CNS pathology in mucopolysaccharidoses

ABSTRACT

The invention provides for nucleotide sequences encoding for a chimeric sulfatase, viral vectors expressing such sequences for gene therapy and pharmaceutical uses of the chimeric expressed protein. The invention is particularly applied in the therapy of mucopolysaccharidosis, preferably type IIIA.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a 371 of PCT/IB2010/056024, filed Dec. 22, 2010, thecontents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to a therapeutic approach, either viralvector-mediated gene therapy or by administration of modifiedsulfatases, in particular the sulfamidase enzyme, to cross theblood-brain barrier and treat the CNS pathology in Mucopolysaccharidoses(MPS), in particular MPS type IIIA.

BACKGROUND OF THE INVENTION

Mucopolysaccharidosis type IIIA (MPS-IIIA) is an inherited diseasecaused by the deficiency of sulfamidase (SGSH), an enzyme involved inthe stepwise degradation of large macromolecules called heparansulfates. As a consequence, undegraded substrates accumulate in thecells and tissues of the affected patients causing cell damage. Thecentral nervous system (CNS) is the predominant target of damage and infact, MPS-IIIA patients show severe mental retardation andneuropathological decline that ultimately leads to death (often<20years). Clinical symptoms include hyperactivity, aggressive behaviourand sleep disturbance (1).

A naturally occurring mouse model of MPS-IIIA has been identified withpathophysiology and symptoms that resemble the human condition (2-4).These mice represent an ideal model to study the physiopathology of thisdisorder and to test new therapeutic protocols.

The treatment of brain lesions represents the principal goal of anytherapeutic approach for MPS-IIIA. A route to reach the brain consistsin the direct injection of a therapeutic molecule directly into thebrain. A number of different enzyme replacement therapy (ERT) protocolshave been tested. In these protocols, a recombinant sulfamidase enzymewas administered through the direct injection into the brain of MPSIIIAmice. These strategies are able to delay the appearance ofneurodegenerative changes when sulfamidase is administered in theyounger mice (5, 6). In addiction, a Gene Therapy protocol based on theintracerebral injection of the SGSH gene via AAV vectors wassuccessfully developed by the authors of the invention (7). Althoughthese direct brain-targeting approaches have been shown to be clinicallyeffective they represent highly invasive approaches for humantherapeutic applicability.

Since every neuron in the brain is perfused by its own blood vessel, aneffective alternative low-invasive route to reach the brain is theintravenous administration of the therapeutic molecule (8). However,this very dense network of microvasculature, which forms the Blood-BrainBarrier (BBB), is not permeable to all the molecules and might impedeeffective delivery of therapeutic agents (9). Indeed, intravenousadministration of lysosomal enzymes has produced a therapeutic effect onthe somatic pathology of many LSDs but it has no or little effect on theCNS pathology due to the impermeability of the BBB to large molecules(10). In MPS-IIIA, it has been demonstrated that intravenous injectionof sulfamidase does not alter the pathology or behavioural processoccurring in the MPS-IIIA mouse brain when the enzyme is supplied afterthe BBB has been formed (11).

Importantly, a recent study by Urayama et al. demonstrated thatsulfamidase is transported across the BBB in neonatal mice throughoutthe mannose 6-phosphate receptor-mediated transport but the influx intoadult brain was negligible (12).

It is clear that in such context the real challenge for the therapy ofMPS-IIIA and in general for all LSDs involving the CNS is to develop CNSsystemic treatment strategies that can overcome the major obstaclerepresented by BBB. An effective strategy to cross the BBB is thetargeting of proteins to the CNS via receptor-mediated transcytosis(13). Well-characterized BBB receptors include: low density lipoproteinreceptor (LDLR), the transferrin receptor (TfR), and the insulin-likegrowth factor receptor (IGF-R). The LDLR family represents a group ofcell surface receptors that binds apolipoprotein (Apo) complexes (lipidcarriers) for the internalizing into the lysosomes (14-16). On thesurface of the BBB, LDLR binding to Apo results in the transcytosis tothe luminal side of the BBB, where the apolipoprotein is released to beuptaken by neurons and astrocytes. A recent study has demonstrated thatfusing the LDLR-binding domain of Apo to a lysosome enzyme results in anefficient delivery of the chimeric enzyme to the CNS (17).

WO2004108071 refers to a chimeric CNS targeting polypeptide comprising aBBB-receptor binding domain, such as the Apolipoprotein B bindingdomain, for therapeutic use in lysosomal storage diseases.

WO2004064750 refers to nucleic acids encoding a chimeric lysosomalpolypeptide (specifically the lysosomal acid glucosidase GAA implicatedin the lysosomal storage disorder Glycogen storage disease type II)comprising a secretory signal sequence (i.e. Vi-antitrypsin andalpha-l-antitrypsin) and the related AAV vectors.

WO2005002515 refers to a compound comprising a megalin-binding moietyconjugated to an agent of interest for receptor mediated drug delivery,particularly by transcytosis, across the blood-brain barrier. Moreoverthe document refers to a method of treating a lysosomal storage diseasebased on the administration of a composition comprising amegalin-binding moiety. Apolipoprotein B and Mucopolysaccharidosis IIIAare mentioned.

WO2009131698 refers to a therapy based on a chimeric NaGlu enzymecharacterized by an Apolipoprotein B binding domain and directedspecifically to Mucopolysaccharidosis IIIB.

Cardone et al. (Hum Mol Gen, 2006 15(7):1225) describes the correctionof Hunter syndrome (the lysosomal storage disease MucopolysaccharidosisType II) in the MPSII mouse model by liver-directed AAV2/8-TBG-mediatedgene delivery.

WO2007092563 refers to a method and compositions for tolerizing amammal's brain to exogenously administered acid sphingomyelinasepolypeptide by first delivering an effective amount of a transgeneencoding the polypeptide to the mammal's hepatic tissue and thenadministering an effective amount of the transgene to the mammal'scentral nervous system (CNS). The therapeutic approach is directed toNiemann-Pick disease, a lysosomal storage disease. Liver- specificpromoters and AAV type 8 are mentioned.

WO2009075815 refers to methods of treating Pompe disease (a lysosomalstorage disease) which involves the administration of an AAV vector inthe context of enzyme replacement therapy. Liver-specific promoter(thyroid hormone-binding globulin promoter) and AAV type 8 arementioned.

None of the above prior art cited documents disclose or even suggest themodified sufamidase enzyme of the instant invention and that it may havea therapeutic effect for the treatment of MPS type IIIA.

SUMMARY OF THE INVENTION

As disclosed in the background art, brain pathology is the most commonfeature in lysosomal storage disorders. Therefore, the treatment ofbrain lesions represents the principal goal of any effective therapy forthese disorders.

The major obstacle to efficiently treat the brain by systemic deliveryof a therapeutic agent is the blood brain barrier (BBB).

Authors developed a new non-invasive therapeutic approach to treat thebrain pathology in the mucopolysaccharidosis type IIIA (MPS-IIIA), alysosomal storage disorder with a severe central nervous systeminvolvement. This strategy is based on the construction of a chimericsulfamidase (the sulfatase enzyme which is deficient in MPS-IIIA),optimized with two amino-acid sequences (one to the N-terminus and theother to the C-terminus of the protein) which confer to the modifiedsulfamidase the capability to be highly secreted and efficientlytargeted to the brain by crossing the blood brain barrier (BBB). Themodified enzyme is expressed by adeno-associated virus (AAV) serotype 8which specifically target the liver and make it like a factory organ ofthe therapeutic enzyme.

The modified sulfamidase may be effectively used for both gene therapyand for enzyme replacement therapy (ERT).

The modification approach may be used for other lysosomal enzymes whichare deficient in other mucopolisaccharidoses with severe CNSinvolvement.

Therefore it is an object of the instant invention a nucleotide sequenceencoding for a chimeric sulfatase, said chimeric sulfatase essentiallyconsisting in the N-terminal-C-terminal sequence order of: a) a signalpeptide derived by either the human α-antitrypsin (hAAT) amino acidsequence or the human Iduronate-2-sulfatase (IDS) amino acid sequence;b) a human sulfatase derived amino acid sequence deprived of its signalpeptide; c) the ApoB LDLR-binding domain.

In a preferred embodiment the encoded signal peptide has a sequencebelonging to the following group: MPSSVSWGILLLAGLCCLVPVSLA (SEQ ID No.2) or MPPPRTGRGLLWLGLVLSSVCVALG (SEQ ID No. 4 or 6).

In a preferred embodiment the nucleotide the human sulfatase is thehuman sulfamidase, more preferably the encoded human sulfamidase derivedamino acid sequence has essentially the sequence:

(SEQ ID No. 8) MSCPVPACCALLLVLGLCRARPRNALLLLADDGGFESGAYNNSAIATPHLDALARRSLLFRNAFTSVSSCSPSRASLLTGLPQHQNGMYGLHQDVHHFNSFDKVRSLPLLLSQAGVRTGIIGKKHVGPETVYPFDFAYTEENGSVLQVGRNITRIKLLVRKFLQTQDDRPFFLYVAFHDPHRCGHSQPQYGTFCEKFGNGESGMGRIPDWTPQAYDPLDVLVPYFVPNTPAARADLAAQYTTVGRMDQGVGLVLQELRDAGVLNDTLVIFTSDNGIPFPSGRTNLYWPGTAEPLLVSSPEHPKRWGQVSEAYVSLLDLTPTILDWFSIPYPSYAIFGSKTIHLTGRSLLPALEAEPLWATVFGSQSHHEVTMSYPMRSVQHRHFRLVHNLNFKMPFPIDQDFYVSPTFQDLLNRTTAGQPTGWYKDLRHYYYRARWELYDRSRDPHETQNLATDPRFAQLLEMLRDQLAKWQWETHDPWVCAPDGVLEEKLSPQCQPLHN EL.Such sequence is encoded by SEQ ID No. 7 nt sequence:

5′- ATGAGCTGCCCCGTGCCCGCCTGCTGCGCGCTGCTGCTAGTCCTGGGGCTCTGCCGGGCGCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGTACAACAACAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTCAGCAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGGATGTACGGGCTGCACCAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGCACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTTTGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGATGACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCCCAGCCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAACGGAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACTGGACGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCGTCGGCCGCATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTGGTGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCACTGCTGAACCCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGACCTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCGAAGACCATCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCACCACGAGGTCACCATGTCCTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCACAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCAGCCCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAGCCGGGACCCCCACGAGACCCAGAACCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGCCAAGTGGCAGTGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCCCAGTGCCAGCCCCTCCACAAT GAGCTGTGA-3′.

In a preferred embodiment the encoded ApoB LDLR-binding domain hasessentially the sequence:

(SEQ ID No. 10) SVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS.

In a preferred embodiment the nucleotide sequence has essentially thesequence belonging to the following group:

SEQUENCES WITH FLAG (expert shall easily substi-tute the flag sequence with any other suitable spacer sequence):a) Assembly hAATsp-SGSH-3xflag cassette (1611). (SEQ ID No. 11) 5′-ATGCCGTCTTCTGTCTCGTGGGGCATCCTCCTGCTGGCAGGCCTGTGCTGCCTGGTCCCTGTCTCCCTGGCTCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGTACAACAACAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTCAGCAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGGATGTACGGGCTGCACCAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGCACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTTTGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGATGACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCCCAACCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAACGGAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACTGGACGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCGTCGGCCGCATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTGGTGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCACTGCTGAACCCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGACCTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCGAAGACCATCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCACCACGAGGTCACCATGTCCTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCACAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCAGCCCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAGCCGGGACCCCCACGAGACCCAGAACCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGCCAAGTGGCAGTGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCCCAGTGCCAGCCCCTCCACAATGAGCTGTCATCTAGAGGATCCCGGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATG ACAAGTAGTGA-3′b) Assembly hIDSsp-SGSH-3xflag cassette (1614 bp). (SEQ ID No. 13) 5′-ATGCCCCCGCCCCGCACCGGCCGCGGCCTGCTGTGGCTGGGCCTGGTGCTGAGCAGCGTGTGCGTGGCCCTGGGCCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGTACAACAACAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTCAGCAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGGATGTACGGGCTGCACCAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGCACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTTTGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGATGACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCCCAACCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAACGGAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACTGGACGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCGTCGGCCGCATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTGGTGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCACTGCTGAACCCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGACCTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCGAAGACCATCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCACCACGAGGTCACCATGTCCTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCACAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCAGCCCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAGCCGGGACCCCCACGAGACCCAGAACCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGCCAAGTGGCAGTGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCCCAGTGCCAGCCCCTACACAATGAGCTCTCATCTAGAGGATCCCGGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACG ATGACAAGTAGTGA-3′c) Assembly hAATsp-SGSH-3xflag-ApoB cassette (1734 bp). (SEQ ID No. 15)5′- ATGCCGTCTTCTGTCTCGTGGGGCATCCTCCTGCTGGCAGGCCTGTGCTGCCTGGTCCCTGTCTCCCTGGCTCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGTACAACAACAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTCAGCAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGGATGTACGGGCTGCACCAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGCACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTTTGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGATGACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCCCAACCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAACGGAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACTGGACGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCGTCGGCCGCATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTGGTGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCACTGCTGAACCCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGACCTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCGAAGACCATCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCACCACGAGGTCACCATGTCTTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCACAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCAGCCCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAGCCGGGACCCCCACGAGACCCAGAACCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGCCAAGTGGCAGTGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCCCAGTGCCAGCCCCTCCACAATGAGCTGTCATCTAGAGGATCCCGGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGATCTCTGTCATTGATGCACTGCAGTACAAATTAGAGGGCACCACAAGATTGACAAGAAAAAGGGGATTGAAGTTAGCCACAGCTCTGTCTCTGAGCAACAAATTTGTGGAGGGTAGTAGATCTTAGTGA-3′d) Assembly hIDSsp-SGSH-3xflag-ApoB cassette (1737 bp). (SEQ ID No. 17)5′- ATGCCCCCGCCCCGCACCGGCCGCGGCCTGCTGTGGCTGGGCCTGGTGCTGAGCAGCGTGTGCGTGGCCCTGGGCCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGTACAACAACAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTCAGCAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGGATGTACGGGCTGCACCAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGCACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTTTGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGATGACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCCCAACCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAACGGAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACTGGACGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCGTCGGCCGCATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTGGTGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCACTGCTGAACCCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGACCTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCGAAGACCATCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCACCACGAGGTCACCATGTCCTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCACAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCAGCCCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAGCCGGGACCCCCACGAGACCCAGAACCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGCCAAGTGGCAGTGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCCCAGTGCCAGCCCCTACACAATGAGCTCTCATCTAGAGGATCCCGGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGATCTCTGTCATTGATGCACTGCAGTACAAATTAGAGGGCACCACAAGATTGACAAGAAAAAGGGGATTGAAGTTAGCCACAGCTCTGTCTCTGAGCAACAAATTTGTGGAGGGTAGTAGATCTTAGTGA-3′ SEQUENCES WITHOUT FLAG:e) Assembly hAATsp-SGSH cassette. (SEQ ID No. 19) 5′-ATGCCGTCTTCTGTCTCGTGGGGCATCCTCCTGCTGGCAGGCCTGTGCTGCCTGGTCCCTGTCTCCCTGGCTCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGTACAACAACAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTCAGCAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGGATGTACGGGCTGCACCAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGCACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTTTGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGATGACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCCCAACCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAACGGAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACTGGACGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCGTCGGCCGCATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTGGTGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCACTGCTGAACCCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGACCTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCGAAGACCATCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCACCACGAGGTCACCATGTCCTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCACAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCAGCCCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAGCCGGGACCCCCACGAGACCCAGAACCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGCCAAGTGGCAGTGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCCCAGTGCCAGCCCCTCCACAATGAGCTGTGA-3′ f) Assembly hIDSsp-SGSH cassette.(SEQ ID No. 21) 5′- ATGCCCCCGCCCCGCACCGGCCGCGGCCTGCTGTGGCTGGGCCTGGTGCTGAGCAGCGTGTGCGTGGCCCTGGGCCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGTACAACAACAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTCAGCAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGGATGTACGGGCTGCACCAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGCACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTTTGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGATGACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCCCAACCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAACGGAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACTGGACGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCGTCGGCCGCATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTGGTGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCACTGCTGAACCCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGACCTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCGAAGACCATCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCACCACGAGGTCACCATGTCCTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCACAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCAGCCCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAGCCGGGACCCCCACGAGACCCAGAACCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGCCAAGTGGCAGTGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCCCAGTGCCAGCCCCTACACAATGAGCTCTGA-3′ g) Assembly hAATsp-SGSH-ApoB cassette.(SEQ ID No. 23) 5′- ATGCCGTCTTCTGTCTCGTGGGGCATCCTCCTGCTGGCAGGCCTGTGCTGCCTGGTCCCTGTCTCCCTGGCTCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGTACAACAACAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTCAGCAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGGATGTACGGGCTGCACCAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGCACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTTTGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGATGACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCCCAACCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAACGGAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACTGGACGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCGTCGGCCGCATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTGGTGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCACTGCTGAACCCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGACCTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCGAAGACCATCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCACCACGAGGTCACCATGTCTTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCACAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCAGCCCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAGCCGGGACCCCCACGAGACCCAGAACCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGCCAAGTGGCAGTGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCCCAGTGCCAGCCCCTCCACAATGAGCTGTCATCTAGATCTGTCATTGATGCACTGCAGTACAAATTAGAGGGCACCACAAGATTGACAAGAAAAAGGGGATTGAAGTTAGCCACAGCTCTGTCTCTGAGCAACAAATTTGTGGAGGGTAGTAGATCTTAG TGA-3′h) Assembly hIDSsp-SGSH-ApoB cassette. (SEQ ID No. 25) 5′-ATGCCCCCGCCCCGCACCGGCCGCGGCCTGCTGTGGCTGGGCCTGGTGCTGAGCAGCGTGTGCGTGGCCCTGGGCCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGTACAACAACAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTCAGCAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGGATGTACGGGCTGCACCAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGCACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTTTGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGATGACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCCCAACCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAACGGAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACTGGACGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCGTCGGCCGCATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTGGTGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCACTGCTGAACCCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGACCTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCGAAGACCATCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCACCACGAGGTCACCATGTCTTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCACAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCAGCCCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAGCCGGGACCCCCACGAGACCCAGAACCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGCCAAGTGGCAGTGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCCCAGTGCCAGCCCCTCCACAATGAGCTGTCATCTAGATCTGTCATTGATGCACTGCAGTACAAATTAGAGGGCACCACAAGATTGACAAGAAAAAGGGGATTGAAGTTAGCCACAGCTCTGTCTCTGAGCAACAAATTTGTGGAGGGTAGTAGATCT TAGTGA-3′.

It is a further object of the invention a recombinant plasmid suitablefor gene therapy of MPS comprising the nucleotide sequence as abovedisclosed under the control of a liver specific promoter, preferably theliver specific promoter is the human thyroid hormone-globulin (TBG)promoter, more preferably the human thyroid hormone-globulin (TBG)promoter has essentially the sequence:

(SEQ ID No. 27) 5′-GCTAGCAGGTTAATTTTTAAAAAGCAGTCAAAAGTCCAAGTGGCCCTTGGCAGCATTTACTCTCTCTGTTTGCTCTGGTTAATAATCTCAGGAGCACAAACATTCCAGATCCAGGTTAATTTTTAAAAAGCAGTCAAAAGTCCAAGTGGCCCTTGGCAGCATTTACTCTCTCTGTTTGCTCTGGTTAATAATCTCAGGAGCACAAACATTCCAGATCCGGCGCGCCAGGGCTGGAAGCTACCTTTGACATCATTTCCTCTGCGAATGCATGTATAATTTCTACAGAACCTATTAGAAAGGATCACCCAGCCTCTGCTTTTGTACAACTTTCCCTTAAAAAACTGCCAATTCCACTGCTGTTTGGCCCAATAGTGAGAACTTTTTCCTGCTGCCTCTTGGTGCTTTTGCCTATGGCCCCTATTCTGCCTGCTGAAGACACTCTTGCCAGCATGGACTTAAACCCCTCCAGCTCTGACAATCCTCTTTCTCTTTTGTTTTACATGAAGGGTCTGGCAGCCAAAGCAATCACTCAAAGTTCAAACCTTATCATTTTTTGCTTTGTTCCTCTTGGCCTTGGTTTTGTACATCAGCTTTGAAAATACCATCCCAGGGTTAATGCTGGGGTTAATTTATAACTAAGAGTGCTCTAGTTTTGCAATACAGGACATGCTATAAAAATGGAAAGATGTTGCTTTCT GAGAGACTGCAG-3′.

The expert in the field will realize that the recombinant plasmid of theinvention has to be assembled in a viral vector for gene therapy oflysosomal disorders, and select the most suitable one. Such viralvectors may belong to the group of: lentiviral vectors, helper-dependentadenoviral vectors or AAV vectors. As example lentiviral vectors forgene therapy of lysosomal storage disorders is described in Naldini, L.,Blomer, U., Gage, F. H., Trono, D., and Verma, I. M. (1996a). In vivogene delivery and stable transduction of nondividing cells by alentiviral vector. Science 272(5259), 263-7; Consiglio A, Quattrini A,Martino S, Bensadoun J C, Dolcetta D, Trojani A, Benaglia G, MarchesiniS, Cestari V, Oliverio A, Bordignon C, Naldini. In vivo gene therapy ofmetachromatic leukodystrophy by lentiviral vectors: correction ofneuropathology and protection against learning impairments in affectedmice L. Nat Med. 2001 March; 7(3):310-6; Follenzi A, Naldini L.HIV-based vectors. Preparation and use. Methods Mol Med. 2002;69:259-74.As a further example helper-dependent adenoviral vectors are describedin Brunetti-Pierri N, Ng P. Progress towards liver and lung-directedgene therapy with helper-dependent adenoviral vectors. Curr Gene Ther.2009 October; 9(5):329-40.

In a preferred embodiment the recombinant plasmid derives from theplasmid vector AAV2.1 and is suitable for AAV viral vectors, preferablyAAV serotype 8.

Then it is a further object of the invention a viral vector for genetherapy of lysosomal disorders comprising any of the recombinant nucleicacid vectors as above disclosed. Preferably the lysosomal disorder isMPS, more preferably MPS type IIIA.

It is a further object of the invention a pharmaceutical compositioncomprising the viral vector as above disclosed, preferably for systemicadministration.

It is a further object of the invention a chimeric sulfatase essentiallyconsisting in the N-terminal-C-terminal sequence order of a) a signalpeptide derived by either the human a-antitrypsin (hAAT) amino acidsequence or the human Iduronate-2-sulfatase (IDS) amino acid sequence;b) an human sulfatase derived amino acid sequence deprived of its signalpeptide; c) the ApoB LDLR-binding domain.

In a preferred embodiment the chimeric sulfatase has a signal peptidehaving a sequence belonging to the following group: (SEQ ID No. 2) or(SEQ ID No. 4).

In a preferred embodiment the chimeric sulfatase has a human sulfamidasederived sequence, preferably (SEQ ID No. 8).

In a preferred embodiment the chimeric sulfatase comprises an encodedApoB LDLR-binding domain having essentially the sequence of (SEQ ID No.10).

In a preferred embodiment the chimeric sulfatase has essentially thesequence belonging to the following group:

SEQUENCE WITH FLAG (expert shall easily substi-tute the flag sequence with any other suitable spacer sequence):a) hAATsp-SGSH-3xflag aminoacid sequence (* = stop). (SEQ ID No. 12)MPSSVSWGILLLAGLCCLVPVSLARPRNALLLLADDGGFESGAYNNSAIATPHLDALARRSLLFRNAFTSVSSCSPSRASLLTGLPQHQNGMYGLHQDVHHFNSFDKVRSLPLLLSQAGVRTGIIGKKHVGPETVYPFDFAYTEENGSVLQVGRNITRIKLLVRKFLQTQDDRPFFLYVAFHDPHRCGHSQPQYGTFCEKFGNGESGMGRIPDWTPQAYDPLDVLVPYFVPNTPAARADLAAQYTTVGRMDQGVGLVLQELRDAGVLNDTLVIFTSDNGIPFPSGRTNLYWPGTAEPLLVSSPEHPKRWGQVSEAYVSLLDLTPTILDWFSIPYPSYAIFGSKTIHLTGRSLLPALEAEPLWATVFGSQSHHEVTMSYPMRSVQHRHFRLVHNLNFKMPFPIDQDFYVSPTFQDLLNRTTAGQPTGWYKDLRHYYYRARWELYDRSRDPHETQNLATDPRFAQLLEMLRDQLAKWQWETHDPWVCAPDGVLEEKLSPQCQPLHNELSSRGSRADYKDHDGDYKDHDIDYKDDDDK**b) hIDSsp-SGSH-3xflag aminoacid sequence (* = stop) (SEQ ID No. 14)MPPPRTGRGLLWLGLVLSSVCVALGRPRNALLLLADDGGFESGAYNNSAIATPHLDALARRSLLFRNAFTSVSSCSPSRASLLTGLPQHQNGMYGLHQDVHHFNSFDKVRSLPLLLSQAGVRTGIIGKKHVGPETVYPFDFAYTEENGSVLQVGRNITRIKLLVRKFLQTQDDRPFFLYVAFHDPHRCGHSQPQYGTFCEKFGNGESGMGRIPDWTPQAYDPLDVLVPYFVPNTPAARADLAAQYTTVGRMDQGVGLVLQELRDAGVLNDTLVIFTSDNGIPFPSGRTNLYWPGTAEPLLVSSPEHPKRWGQVSEAYVSLLDLTPTILDWFSIPYPSYAIFGSKTIHLTGRSLLPALEAEPLWATVFGSQSHHEVTMSYPMRSVQHRHFRLVHNLNFKMPFPIDQDFYVSPTFQDLLNRTTAGQPTGWYKDLRHYYYRARWELYDRSRDPHETQNLATDPRFAQLLEMLRDQLAKWQWETHDPWVCAPDGVLEEKLSPQCQPLHNELSSRGSRADYKDHDGDYKDHDIDYKDDDDK**c) hAATsp-SGSH-3xflag-ApoB aminoacid sequence (* = stop) (SEQ ID No. 16)MPSSVSWGILLLAGLCCLVPVSLARPRNALLLLADDGGFESGAYNNSAIATPHLDALARRSLLFRNAFTSVSSCSPSRASLLTGLPQHQNGMYGLHQDVHHFNSFDKVRSLPLLLSQAGVRTGIIGKKHVGPETVYPFDFAYTEENGSVLQVGRNITRIKLLVRKFLQTQDDRPFFLYVAFHDPHRCGHSQPQYGTFCEKFGNGESGMGRIPDWTPQAYDPLDVLVPYFVPNTPAARADLAAQYTTVGRMDQGVGLVLQELRDAGVLNDTLVIFTSDNGIPFPSGRTNLYWPGTAEPLLVSSPEHPKRWGQVSEAYVSLLDLTPTILDWFSIPYPSYAIFGSKTIHLTGRSLLPALEAEPLWATVFGSQSHHEVTMSYPMRSVQHRHFRLVHNLNFKMPFPIDQDFYVSPTFQDLLNRTTAGQPTGWYKDLRHYYYRARWELYDRSRDPHETQNLATDPRFAQLLEMLRDQLAKWQWETHDPWVCAPDGVLEEKLSPQCQPLHNELSSRGSRADYKDHDGDYKDHDIDYKDDDDKISVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSRS**d) hIDSsp-SGSH-3xflag-ApoB aminoacid sequence (* = stop) (SEQ ID No. 18)MPPPRTGRGLLWLGLVLSSVCVALGRPRNALLLLADDGGFESGAYNNSAIATPHLDALARRSLLFRNAFTSVSSCSPSRASLLTGLPQHQNGMYGLHQDVHHFNSFDKVRSLPLLLSQAGVRTGIIGKKHVGPETVYPFDFAYTEENGSVLQVGRNITRIKLLVRKFLQTQDDRPFFLYVAFHDPHRCGHSQPQYGTFCEKFGNGESGMGRIPDWTPQAYDPLDVLVPYFVPNTPAARADLAAQYTTVGRMDQGVGLVLQELRDAGVLNDTLVIFTSDNGIPFPSGRTNLYWPGTAEPLLVSSPEHPKRWGQVSEAYVSLLDLTPTILDWFSIPYPSYAIFGSKTIHLTGRSLLPALEAEPLWATVFGSQSHHEVTMSYPMRSVQHRHFRLVHNLNFKMPFPIDQDFYVSPTFQDLLNRTTAGQPTGWYKDLRHYYYRARWELYDRSRDPHETQNLATDPRFAQLLEMLRDQLAKWQWETHDPWVCAPDGVLEEKLSPQCQPLHNELSSRGSRADYKDHDGDYKDHDIDYKDDDDKISVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSRS**, SEQUENCES WITHOUT FLAG:e) hAATsp-SGSH aminoacid sequence (* = stop) (SEQ ID No. 20)MPSSVSWGILLLAGLCCLVPVSLARPRNALLLLADDGGFESGAYNNSAIATPHLDALARRSLLFRNAFTSVSSCSPSRASLLTGLPQHQNGMYGLHQDVHHFNSFDKVRSLPLLLSQAGVRTGIIGKKHVGPETVYPFDFAYTEENGSVLQVGRNITRIKLLVRKFLQTQDDRPFFLYVAFHDPHRCGHSQPQYGTFCEKFGNGESGMGRIPDWTPQAYDPLDVLVPYFVPNTPAARADLAAQYTTVGRMDQGVGLVLQELRDAGVLNDTLVIFTSDNGIPFPSGRTNLYWPGTAEPLLVSSPEHPKRWGQVSEAYVSLLDLTPTILDWFSIPYPSYAIFGSKTIHLTGRSLLPALEAEPLWATVFGSQSHHEVTMSYPMRSVQHRHFRLVHNLNFKMPFPIDQDFYVSPTFQDLLNRTTAGQPTGWYKDLRHYYYRARWELYDRSRDPHETQNLATDPRFAQLLEMLRDQLAKWQWETHDPWVCAPDGVLEEKLSPQCQ PLHNEL*f) hIDSsp-SGSH aminoacid sequence (* = stop) (SEQ ID No. 22)MPPPRTGRGLLWLGLVLSSVCVALGRPRNALLLLADDGGFESGAYNNSAIATPHLDALARRSLLFRNAFTSVSSCSPSRASLLTGLPQHQNGMYGLHQDVHHFNSFDKVRSLPLLLSQAGVRTGIIGKKHVGPETVYPFDFAYTEENGSVLQVGRNITRIKLLVRKFLQTQDDRPFFLYVAFHDPHRCGHSQPQYGTFCEKFGNGESGMGRIPDWTPQAYDPLDVLVPYFVPNTPAARADLAAQYTTVGRMDQGVGLVLQELRDAGVLNDTLVIFTSDNGIPFPSGRTNLYWPGTAEPLLVSSPEHPKRWGQVSEAYVSLLDLTPTILDWFSIPYPSYAIFGSKTIHLTGRSLLPALEAEPLWATVFGSQSHHEVTMSYPMRSVQHRHFRLVHNLNFKMPFPIDQDFYVSPTFQDLLNRTTAGQPTGWYKDLRHYYYRARWELYDRSRDPHETQNLATDPRFAQLLEMLRDQLAKWQWETHDPWVCAPDGVLEEKLSPQC QPLHNEL*g) hAATsp-SGSH-ApoB aminoacid sequence (* = stop) (SEQ ID No. 24)MPSSVSWGILLLAGLCCLVPVSLARPRNALLLLADDGGFESGAYNNSAIATPHLDALARRSLLFRNAFTSVSSCSPSRASLLTGLPQHQNGMYGLHQDVHHFNSFDKVRSLPLLLSQAGVRTGIIGKKHVGPETVYPFDFAYTEENGSVLQVGRNITRIKLLVRKFLQTQDDRPFFLYVAFHDPHRCGHSQPQYGTFCEKFGNGESGMGRIPDWTPQAYDPLDVLVPYFVPNTPAARADLAAQYTTVGRMDQGVGLVLQELRDAGVLNDTLVIFTSDNGIPFPSGRTNLYWPGTAEPLLVSSPEHPKRWGQVSEAYVSLLDLTPTILDWFSIPYPSYAIFGSKTIHLTGRSLLPALEAEPLWATVFGSQSHHEVTMSYPMRSVQHRHFRLVHNLNFKMPFPIDQDFYVSPTFQDLLNRTTAGQPTGWYKDLRHYYYRARWELYDRSRDPHETQNLATDPRFAQLLEMLRDQLAKWQWETHDPWVCAPDGVLEEKLSPQCQPLHNELSSRSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSRS* *h) hIDSsp-SGSH-ApoB aminoacid sequence (* = stop) (SEQ ID No. 26)MPPPRTGRGLLWLGLVLSSVCVALGRPRNALLLLADDGGFESGAYNNSAIATPHLDALARRSLLFRNAFTSVSSCSPSRASLLTGLPQHQNGMYGLHQDVHHFNSFDKVRSLPLLLSQAGVRTGIIGKKHVGPETVYPFDFAYTEENGSVLQVGRNITRIKLLVRKFLQTQDDRPFFLYVAFHDPHRCGHSQPQYGTFCEKFGNGESGMGRIPDWTPQAYDPLDVLVPYFVPNTPAARADLAAQYTTVGRMDQGVGLVLQELRDAGVLNDTLVIFTSDNGIPFPSGRTNLYWPGTAEPLLVSSPEHPKRWGQVSEAYVSLLDLTPTILDWFSIPYPSYAIFGSKTIHLTGRSLLPALEAEPLWATVFGSQSHHEVTMSYPMRSVQHRHFRLVHNLNFKMPFPIDQDFYVSPTFQDLLNRTTAGQPTGWYKDLRHYYYRARWELYDRSRDPHETQNLATDPRFAQLLEMLRDQLAKWQWETHDPWVCAPDGVLEEKLSPQCQPLHNELSSRSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSRS **

It is another object of the invention the chimeric sulfatase as abovedisclosed for medical use, preferably for the treatment of MPS, morepreferably MPS type IIIA.

It is another object of the invention a pharmaceutical compositioncomprising the chimeric sulfatase as above disclosed and suitablediluents and/or eccipients and/or carriers.

It is another object of the invention a method for treatment of a MPSpathology comprising the step of administering to a subject a suitableamount of the pharmaceutical composition comprising the viral vector forgene therapy as above disclosed. Preferably the MPS pathology is MPStype IIIA.

It is another object of the invention a method for treatment of a MPSpathology comprising the step of administering to a subject a suitableamount of the pharmaceutical composition comprising the chimericsulfatase as above disclosed. Preferably the MPS pathology is MPS typeIIIA.

Major advantage of the invention is that the chimeric molecule of theinvention as produced and secreted by the liver is able to cross the BBBand thus potentially target to all brain districts.

Regarding the gene therapy approach, with respect to prior art Fraldi etal. HMG 2007 that describes AAV2/5 mediated gene therapy for MPS-IIIIA,the instant invention is less invasive because AAV8 vectors areadministered systemically and not directly into the brain.

As to the enzyme replacement therapy approach with respect to the priorart Hemsley, K. M. and J. J. Hopwood, Behav Brain Res, 2005; Savas, P. Set al., Mol Genet Metab, 2004 and Hemsley, K. M., et al., Mol GenetMetab, 2007, the instant invention overcomes the necessity to repeat theinjection of the enzyme and it is designed to cross the BBB. It is worthto point out that for ERT approaches the BBB and the high cost of theenzyme production are very important limitations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Non-modified SGSH: Preliminary in vivo study 1 (newborntreatment). Analysis of GFP signal in liver of newborn MPSIIA miceinjected with AAV2/8-TBG-GFP. Newborn MPSIIIA were injected withAAV2/8-TBG-SGSH vectors (expressing a not-modified sulfamidase). Ascontrol, newborn MPSIIIA and Heterozygous (phenotypically normal) micewere injected with AAV2/8-TBG-GFP vectors. Liver sections from MPS-IIIAinjected mice were analyzed for GFP staining at different time afterinjection (1,2,3,5 and 10 weeks after injection). The GFP signal wasvery strong at early time points. However, a significant decrease of GFPsignal was observed at later time point after injection

FIG. 2. Non-modified SGSH: Preliminary in vivo study 1 (newborntreatment). SGSH activity in the liver and serum of newborn injectedmice. The sulfamidase activity was measured in the serum (A) and liver(B) of MPSIIIA mice injected with AAV2/8-TBG-SGSH and control mice(MPS-IIIA and heterozygous mice injected with AAV2/8-TBG-GFP). (A) TheSGSH activity in plasma of AAV2/8-TBG-SGSH-treated MPS-IIIA miceincreased during the first two weeks period after neonatal treatment,and then decreased through the time to reach the levels measured incontrol GFP-injected MPS-IIIA mice. (B) The analysis of liver SGSHactivity showed a trend similar to that observed in the plasma withhigher levels of activity detected within the first week afterinjection.

FIG. 3. Non-modified SGSH: Preliminary in vivo study 2 (adulttreatment). Analysis of GFP signal in liver of adult MPSIIA miceinjected with AAV2/8-TBG-GFP. 1.5 months old MPSIIIA were injected withAAV2/8-TBG-SGSH vectors (expressing a not-modified sulfamidase). Ascontrol, 1.5 months old MPSIIIA and Heterozygous (phenotypically normal)mice were injected with AAV2/8-TBG-GFP vectors. Liver sections fromMPS-IIIA injected mice were analyzed for GFP staining at 1 and 5 weeksafter injection. A high and stable expression of the GFP was observed.

FIG. 4. Non-modified SGSH: Preliminary in vivo study 2 (adulttreatment). SGSH activity in the serum and liver of adult injected mice.The sulfamidase activity was measured in the serum (A) and liver (B) ofMPSIIIA mice injected with AAV2/8-TBG-SGSH and control mice (MPS-IIIAand heterozygous mice injected with AAV2/8-TBG-GFP). (A) In the liver ofMPSIIIA mice injected with AAV2/8-TBG-SGSH a strong increase in the SGSHactivity was observed compared to the low enzyme activity detected inthe animals injected with GFP vector. In addition, this activityremained stable for 5 weeks after injection (the last time pointanalyzed). (B)

Consistently, the analysis of SGSH activity in the serum of MPS-IIIAmice treated with AAV2/8-TBG-SGSH was very high and stable duringthroughout the analyzed post-injection time.

FIG. 5. Chimeric sulfamidase constructs. The signal peptide (SP) ofsulfamidase was replaced with that of either human a-antitrypsin (hAAT)or Iduronate-2-sulfatase (IDS). The constructs were designed as“partially engineered sulfamidase proteins” (IDSsp-SGSHflag andhAATsp-SGSHflag). To build the final chimeric sulfamidase proteins, theApoB LDLR-binding domain (ApoB-BD) was fused at the C-terminus of theFlag tag to obtain the resulting “finally engineered constructs”(IDSsp-SGSHflag-ApoB and hAATsp-SGSHflag-ApoB). The ApoB sequence (114bp) was amplified by PCR from the human blood cDNA using forward andreverse oligonucleotides with 5′ BglII sites. The backbone plasmidcontaining the SP-SGSH sequence was prepared inserting by mutagenesisthe BglII site before the stop codon of Flag tag. All the resultingchimeric sulfamidase sequences (IDSsp-SGSHflag, hAATsp-SGSHflag,IDSsp-SGSHflag-ApoB and hAATsp-SGSHflag-ApoB) were inserted in mammalianexpression plasmids under a CMV promoter.

FIG. 6. Receptor-mediated transport. Crossing the BBB viareceptor-mediated transcytosis. The Low Density Lipoprotein receptor(LDLR)-binding domain of the Apolipoprotein B (ApoB LDLR-BD) confers tothe sulfamidase the capability to reach the brain cells by binding LDLreceptors, which are abundant on the endothelial cells of BBB. Thismechanism may substitute the mannose-6-phosphate receptor(M6PR)-mediated transport of the sulfamidase throughout the BBB, whichis inefficient.

FIG. 7. In vitro study. SGSH activity in the pellet and in the medium oftransfected MPS-IIIA MEF cells. MEF cells derived from MPS-IIIA micewere transfected with either partially or finally engineered constructs.(A) The activity of sulfamidase was measured in the medium (light grey)and in the pellet (dark grey) of transfected cells. (B) Thecorresponding efficiency of secretion (activity in medium/totalactivity) was also evaluated.

FIG. 8. In vitro study. Western blot analysis of all engineeredsulfamidase proteins. MEF cells derived from MPS-IIIA mice weretransfected with either partial or final engineered constructs or withcontrol SGSH not modified construct. (A) blot analysis with anti-flagantibodies showing the correct expression of all the chimeric proteins.As a control of transfection efficiency the cells were co-transfectedwith the same concentration of a plasmid containing flag-taggedSyntaxin7, an unrelated protein. (B) Pulse and chase experiments wereperformed in the transfected cells to evaluate the turnover rate of thechimeric proteins (C) Cos-7 cells were transfected with either partiallyor finally engineered constructs or with control SGSH non modifiedconstruct. Lysosomal localization were observed in all transfected cellsby immunostaining with anti-SGSH antibodies.

FIG. 9. In vivo study. Preliminary in vivo results in MPS IIIA miceinjected with finally engineered sulfamidase. Authors obtainedpreliminary but extremely encouraging results in MPS-IIIA mice injectedwith one of the final sulfamidase constructs: hAATsp-SGSHflag-ApoB.Adult MPS-IIIA mice were systemically injected with AAV2/8-TBG-hAATsp-SGSHflag-ApoB. A group of MPS-IIIA were also injected withAAV2/8-TBG-SGSH (containing the non-modified sulfamidase) as control.The mice were sacrificed one month after injection. In the mice injectedwith the chimeric sulfamidase we observed higher liver sulfamidaseactivity and a very strong increase in the sulfamidase secretion withrespect to control mice. Moreover, we detected a significant increase inSGSH activity into the brain of mice injected with the chimericsulfamidase compared to SGSH activity measures in the brain of miceinjected with not-modified sulfamidase.

FIG. 10. Map of AAV2.1 plasmid. Map of pAAV2.1 plasmid used for AAV2.8viral vectors production. The plasmid contains the GFP gene under thecontrol of the liver specific promoter TBG. The GFP sequence wasreplaced with the cDNAs coding the chimeric sulfamidase cassettes byusing NotI and HindIII restriction sites. The resulting plasmid wastransfected along with pAd helper, pAAV rep-cap plasmid in 293 cells toproduce AAV2.8 viral vectors (see Methods).

DETAILED DESCRIPTION OF THE INVENTION

Methods

Construction of Chimeric SGSH Cassettes, Recombinant Nucleic AcidVectors and Viral Vectors

The alternative signal peptides were produced by ligation of twofragments: a sequence from human SGSH cDNA (fragment I) and the signalpeptide sequence (fragment II). Fragment I was amplified from a hSGSHexpressing plasmid and started at the 3′ terminus of hSGSH signalpeptide sequence (corresponding to the nucleotide in position 61 on theSGSH sequence) and extended to a unique XbaI site and contained theentire SGSH cDNA (oligos used: SGSHFOR 5′-CGT CCC CGG AAC GCA CTG CTGCTC CT-3′ (SEQ ID No. 28) and SGSHREV 5′-GCG GCC TCT AGA TGA CAG CTC ATTGTG GAG GGG CTG-3′ (SEQ ID No. 29)). Fragment II was unique for eachexpression cassette. For hAATsp-SGSH-cFlag, fragment II was synthesizedby annealing two specific oligonucleotide sequences (hAATspFOR 5′-GGCCGC ATG CCG TCT TCT GTC TCG TGG GGC ATC CTC CTG CTG GCA GGC CTG TGC TGCCTG GTC CCT GTC TCC CTG GCT 3′ (SEQ ID No. 30) and hAATspREV 5′-AGC CAGGGA GAC AGG GAC CAG GCA GCA CAG GCC TGC CAG CAG GAG GAT GCC CCACGA GACAGA AGA CGG CAT GC-3′ (SEQ ID No. 31)) containing the humanα1-antitrypsin signal peptide sequence [human a1-antitrypsin cDNA: 72bp].

The fragment encoding for such signal peptide was:

(SEQ ID No. 1) 5′-ATGCCGTCTTCTGTCTCGTGGGGCATCCTCCTGCTGGCAGGCCTGTGCTGCCTGGTCCCTGTCTCCCTGGCT-3′.

For IDSsp-SGSH-cFlag expression cassette, fragment II was synthesized byannealing two specific oligonucleotide sequences (IDSspFOR 5′-GGC CGCATG CCC CCG CCC CGC ACC GGC CGC GGC CTG CTG TGG CTG GGC CTG GTG CTG AGCAGC GTG TGC GTG GCC CTG GGC-3′ (SEQ ID No. 32) and IDSspREV 5′-GCC CAGGGC CAC GCA CAC GCT GCT CAG CAC CAG GCC CAG CCA CAG CAG GCC GCG GCC GGTGCG GGG CGG GGG CAT GC-3′ (SEQ ID No. 33) containing the human Iduronatesulfatase signal peptide sequence [Homo sapiens iduronate 2-sulfatase(IDS) cDNA: 75 bp]. The fragment encoding for such signal peptide was:5′-ATGCCGCCACCCCGGACCGGCCGAGGCCTTCTCTGGCTGGGTCTGGTTCTGAGCTCCGTCTGCGTCGCCCTCGGA-3′ (SEQ ID No. 3) or an optimized sequenze5′-ATGCCCCCGCCCCGCACCGGCCGCGGCCTGCTGTGGCTGGGCCTGGTGCTGAGCAGCGTGTGCGTGGCCCTGGGC-3′ (SEQ ID No. 5). The two above sequencesdiffer only for the codon usage and encode for the same signal peptideaa. sequence (SEQ ID No. 4 or 6). The oligonucleotide sequences offragment II have 5′ NotI site and 3′ blunt end site. The forward andreverse oligonucleotide sequences were incubated for three minutes at100° C. After chilling at RT we added the PNK to oligos for 30 minutesat 37° C. The fragment I (5′NotI-3′blunt) and fragment II(5′blunt-3′Xba) were ligated with p3×Flag-CMV14 vector plasmid(5′Not-3′Xba). DH5α competent cells was transformed with the resultingligation mix.

To obtain the complete SGSH chimeric constructs, the amino acid sequence3371-3409 of human ApoB (114 bp: 5′TCTGTCATTGATGCACTGCAGTACAAATTAGAGGGCACCACAAGATTGACAAGAAAAAGGGGATTGAAGTTAGCCACAGCTCTGTCTCTGAGCAACAAATTTGTGGAGGGTAGT-3′ (SEQ ID No. 9) was amplified by a humancDNA library (oligos: ApoBDFOR 5′-AGA TCT CTG TCA TTG ATG CAC TGC AGT-3′(SEQ ID No. 34) and ApoBDREV 5′-AGA TCT ACT ACC CTC CAC AAA TTT GTTGC-3′(SEQ ID No. 35)) and cloned into the BglII sites at 5′ terminus of3×Flag tag of either hAATsp-SGSH-cFlag or IDSsp-SGSH-cFlag.

The different expression cassettes containing either the partialchimeric constructs (hAATsp-SGSH-cFlag and hIDSsp-SGSH-cFlag) or thecomplete chimeric constructs (hAATsp-SGSH-cFlag-ApoB andhIDSsp-SGSH-cFlag-ApoB) were subcloned in the pAAV2.1-TBG-GFP betweenNotI (5′) and HindIII (3′) (the GFP sequence was replaced with theexpression cassettes). The resulting plasmids (FIG. 10) were used toproduce recombinant AAV serotype 8 (AAV2/8) (19). The AAV vectors wereproduced using a transient transfection of three plasmids in 293 cells:pAd helper, pAAV rep-cap (packaging plasmid containing the AAV2 rep genefused with cap genes of AAV serotype 8), pAAV Cis (this plasmid ispAAV2.1-TGB vector expressing the chimeric sulfamidase proteins). Therecombinant AAV2/8 viral vectors were purified by two rounds of CsCl, asdescribed previously (19). Vector titers, expressed as genome copies(GC/ml), were assessed by real-time PCR (GeneAmp 7000 AppliedBiosystem). The AAV vectors were produced by the TIGEM AAV Vector CoreFacility(http://www.tigem.it/core-facilities/adeno-associated-virus-aav-vector-core).

Trasfections and Secretions in Cells.

Hela and MPSIIIA MEF Cells were maintained in DMEM supplemented with 10%FBS and penicillin/streptomycin (normal culture medium). Sub-confluentcells were transfected using Lipofectamine™ 2000 (Invitrogen) accordingto manufacturer's protocols. One day after transfection the medium wasreplaced with DMEM 0.5% FBS. Two days after transfection we collectedthe conditioned medium and the pellet for the enzyme assays and westernblot analysis.

WB Analysis

3×flag Lysis buffer 1× (50 mM Tris-HCl pH8, 200 mM NaCl, 1% Triton X100,1 mM EDTA, 50 mM HEPES) was added to the cell pellets. The lysates wereobtained by incubating the cell pellets with lysis buffer for 1 hour inice. Protein concentration was determined using the Bio-Rad (Bio-Rad,Hercules, Calif., USA) colorimetric assay. The conditioned medium wasconcentrated in the vivaspin 500 (Sartorius) by centrifugation of themedium at 13.000 rpm for 7 min. Flagged sulfamidase proteins wererevealed by Western Blot analysis using a anti-FLAG M2 monoclonalperoxidase-conjugate antibodies (A8592 Sigma-Aldrich) diluted 1:1000 in5% milk.

Immunofluorescence

Cells were washed three times in cold PBS and then fixed in 4%paraformaldehyde (PFA) for 15 min. Fixed cells were washed four times incold PBS, permeabilized with blocking solution (0.1% Saponin and 10% FBSin PBS) for 30 min and immunolabelled with appropriate primary antibody:Rabbit anti h-sulfamidase (1:300, Sigma). After four washes in PBS weincubated the cells with secondary antibody Anti-Rabbit Alexa fluor-488conjugated (1:1000). Cells were then washed four times in cold PBS andmounted in Vectashield mounting medium.

Pulse and Chase

To determine degradation rates of sulfamidase enzyme, MPSIIIA MEFstransfected with different chimeric constructs were radiolabeled with 30μCi/10⁶ cells [35S]methionine:cysteine mixture (EasyTag™ EXPRE35S35SProtein Labeling Mix, [3S]; PerkinElmer) for 30 minutes inmethionine:cysteine-free medium (Sigma) supplemented with 1% fetal calfserum. After extensive washing, cells were maintained in the presence of5% fetal calf serum and supplemented with methionine and cysteine. Cellswere recovered at different time points and lysed using 3×flag Lysisbuffer. Lysates were cleared by centrifugation and supernatants wereimmunoprecipitated by using agarose-conjugated antibody against flag(anti-flag M2 affinity Gel, A2220Sigma-Aldrich). After extensive washingwith lysis buffer, the immunoprecipitate was subjected to SDS-PAGE.Dried gels were exposed to a PhosphorImager screen and quantified with aPhosphorImager system.

Animals

Homozygous mutant (MPS-IIIA, −/−) and heterozygous (phenotypicallynormal +/−) C57BL/6 mice were utilized. Consequently, the term ‘normalmice’ is used to refer to the mouse phenotype. Experiments wereconducted in accordance with the guidelines of the Animal Care and UseCommittee of Cardarelli Hospital in Naples and authorized by the ItalianMinistry of Health.

Systemic Injection and Tissues Collection

Newborn MPS-IIIA and normal mice at postnatal day 0-1 werecryo-anesthetized. The vectors were delivered in the systemic route viatemporal vein (2×10¹¹ particles in 100 μl). The adult MPSIIIA mice (1month) were injected via caudal vein (2×10¹¹ particles in 100 μl). Theserum of animals were collected at at different time points afterinjection for the enzyme assays. To evaluate liver and braintransduction the animals were sacrificed at different time points. Someof them were perfused/fixed with 4% (w/v) paraformaldehyde in PBS, theliver was then removed for GFP staining. The remaining mice weresacrificed and liver and brain removed to measure SGSH activity.

SGSH Activity Assay

SGSH activity was measured following protocols described in Fraldi etal., Hum Mol Gen 2007).

GFP Analysis

Liver tissues were subjected to a saccharose gradient (from 10 to 30%)and incubated O/N in 30% saccharose at 4° C. Finally, tissues wereembedded in OCT embedding matrix (Kaltek) and snap-frozen in a bath ofdry ice and ethanol. Tissue cryosections were cut at 10 μm of thickness,washed with PBS for 10 min, mounted in Vectashield mounting medium andprocessed for GFP analysis.

Results

The aim of the project was to develop a low-invasive systemic genetherapy strategy based on the intravenous injection of AAV serotype 8.This serotype displays high tropism to the liver (18-20) and can be usedto delivery of an engineered gene encoding a chimeric modifiedsulfamidase optimized (i) to be highly secreted from the liver thusreaching high levels of circulating enzyme in the blood stream.Sulfamidase is poor secreted respect to other sulfatase enzymes such asthe iduronate-2-sulfatase (IDS). Sulfamidase signal peptide was replacedwith that of either IDS or human α-antitrypsin (AAT), a highly secretedenzyme; (ii) to efficiently cross the BBB. The chimeric sulfamidase wasfurther engineered with a specific brain-targeting protein domain, the(LDLR)-binding domain of the apolipoprotein B (ApoB LDLR-BD).

In Vivo Results in MPS IHA Mice

The efficacy of the new treatment is strictly dependent on the abilityof the liver to be highly transduced by the transgene in order toefficiently secrete in the blood stream the sulfamidase that will thencross the BBB and transduce the brain by means of its brain-targetsequence. Therefore, the serum levels of the therapeutic enzyme mayrepresent critical factor in determining the efficacy of the therapy. Noprevious studies have been done to analyze liver transduction and thesystemic levels of SGSH upon systemic gene delivery of exogenous SGSH inMPS-IIIA mice. Thus, we decided to investigate this issue in order toproduce useful preliminary data for designing an effective therapeuticstrategy.

The delivery of therapeutic enzyme to neonatal mice is a useful tool toprevent pathology in MPS-IIIA mice. We then decided to test whether theAAV2/8-mediated systemic injection in newborn MPSIIIA could be afeasible approach to develop our new therapeutic strategy. To this aimwe injected MPS-IIIA newborn mice with AAV2/8 containing the sulfamidasecoding sequence under the control of a liver specific promoter (Thyroidhormone-globulin, TBG) in order to specifically target the liver andmake it like a factory organ of the therapeutic enzyme. Mice wereinjected via temporal vein with 1×10¹¹ particles of virus. Threeexperimental groups of mice were established: control mice (heterozygousmice; these mice display a normal phenotype) treated withAAV2/8-TBG-GFP, MPS-IIIA mice treated with AAV2/8-TBG-GFP and MPS-IIIAmice treated with AAV2/8-TBG-SGSH.

To test the efficiency of injection we analyzed the GFP fluorescence inthe liver of GFP-injected mice (normal and MPS-IIIA mice). The GFPsignal was present at either early or late time point after injection;however, a significant decrease of GFP signal was observed in the liverof mice analyzed at later time point after injection (FIG. 1). TheMPS-IIIA mice injected with AAV2/8-TBG-SGSH were checked for SGSHactivity in plasma and in the liver at different time points afterinjection (5, 8, 10, 14 days and at 3, 4, 5, and 10 weeks). The SGSHactivity in plasma of AAV2/8-TBG-SGSH-treated MPS-IIIA mice increasedduring the first two weeks period after neonatal treatment, and thendecreased through the time to reach the levels measured in controlGFP-injected MPS-IIIA mice (FIG. 2A). The analysis of liver SGSHactivity showed a trend similar to that observed in the plasma withhigher levels of activity detected within the first week after injection(FIG. 2B).

This preliminary study in newborn mice demonstrated that although theliver is efficiently transduced by AAV2/8-mediated neonatal delivery ofsulfamidase, the enzyme is present at low levels (comparable to controlGFP-injected MPS-IIIA mice) into both the liver and serum after 1 weekpost-injection making this approach unfeasible to treat the brain.

To evaluate whether the proliferation of hepatocytes during the periodafter the treatment is responsible for the liver dilution of vectorafter neonatal injection we performed a new study based on the systemic(caudal vein injection) AAV2/8-mediated delivery of SGSH in adult mice(1.5 month of age), in which the liver has completed its growth.

Also in this study we established three experimental groups of mice:normal mice treated with AAV2/8-TBG-GFP, MPS-IIIA mice treated withAAV2/8-TBG-GFP and MPS-IIIA mice treated with AAV2/8-TBG-SGSH. Theanalysis of GFP expression, at different time points after treatment (1week and 5 weeks after injection) underlined a high and stableexpression of the transgene in the liver of adult treated mice (FIG. 3).MPSIIIA treated mice were also checked for the SGSH activity in theliver and in the serum at different time points (1 week, 2-,3-,4-,5-weeks) after the treatment. In the liver of MPSIIIA mice injected withAAV2/8-TBG-SGSH we observed a strong increase of SGSH activity comparedwith low enzyme activity in the animals injected with GFP vector, andthis activity remained stable until 5 weeks after injection (the latertime point analyzed) (FIG. 4A). Also the analysis of SGSH activity inthe serum of treated mice was very high and stable until during theentire post-injection period analyzed (FIG. 4B). Importantly, thistreatment did not result in any detectable sulfamidase activity into thebrain of AAV2/8-injected MPS-IIIA mice (not shown).

In conclusion these preliminary studies show that: (i) liver is highlytransduced by AAV2/8-mediated systemic injection (ii) the decrease ofSGSH activity in the newborn treated mice was due to the dilution ofvector in the liver and allow us to consider the adult mice a good modelto test the systemic treatment with AAV2/8 containing the chimericsulfamidase (iii) the secreted (non modified) sulfamidase did not resultin a detectable enzymatic activity into the brain. The latter is anexpected result and further justifies the rationale behind the aim ofour project.

Construction and Validation of the Chimeric Sulfamidase Proteins

In order to increase sulfamidase secretion from the liver and thus theamount of the enzyme in the blood stream available to specificallytarget the brain, we engineered the sulfamidase by replacing its ownsignal peptide (SP) with an alternative one. Two signal peptides havebeen tested, the Iduronate-2-sulfatase (IDS) signal peptide and thehuman a-antitrypsin (AAT) signal peptide (FIG. 5). The rationale behindthe use of these two signal peptides is that IDS is a lysosomal enzymethat was demonstrated to be secreted at high levels from the liver [21]while the AAT is a highly secreted enzyme. The final goal of our projectis to produce a modified sulfamidase capable to cross the BBB and targetthe CNS via receptor-mediated transcytosis (FIG. 6). For this reasonbefore starting the experiments aimed at evaluating the therapeuticefficacy of the substituting

SP signal in SGSH, we further engineered the modified SGSH with aspecific brain-targeting protein domain, the Low Density Lipoproteinreceptor (LDLR)-binding domain of the Apolipoprotein B (ApoB LDLR-BD).The Binding Domain of ApoB will allow the sulfamidase to reach the braincells by binding LDL receptors, which are abundant on the endothelialcells of BBB (FIG. 6). The two finally engineered sulfamidase constructscontain at C-terminal the ApoB LDLR-BD and at N-terminal either an IDSor an hAAT signal peptide (IDSsp-SGSHflag-ApoB and hAATsp-SGSHflag-ApoB)(FIG. 5).

To evaluate the functionality of chimeric sulfamidase proteins wetransfected MPSIIIA MEF cells with either partial or final engineeredsulfamidase proteins and compared the outcomes with those resulting fromthe transfections with not-engineered sulfamidase. Surprisingly, weobserved that SGSH activity in the pellet and in the conditioned mediumwas higher in the cells transfected with the final chimeric constructscompared with the activity measured in the cells transfected with theother constructs, indicating that finally engineered sulfamidase wereefficiently secreted (FIG. 7A). Indeed, these results were associatedwith a higher secretion efficiency of the finally engineered sulfamidaseenzymes with respect to non-engineered sulfamidase (FIG. 7B). However,this secretion efficiency was similar to that measured aftertransfection of partially chimeric sulfamidase (containing only thealternative signal peptide) (FIG. 7B). Remarkably, we observed that themodifications of the sulfamidase, in particular those present in thefinally engineered sulfamidase, confer to the chimeric proteins a higherstability compared to the non-engineered sulfamidase (FIGS. 8A and B).Thus, we concluded that the increase in the sulfamidase protein levelsin the medium of cells transfected with engineered sulfamidase proteinswas due to both increased efficiency in secretion and increasedstability of engineered sulfamidase.

Moreover, immunostaining with anti-SGSH antibodies showed alysosomal-like localization for both partial and final engineeredconstructs (FIG. 8C).

In conclusion these results demonstrate that: (i) the chimericsulfamidase enzymes containing the alternative signal peptide arefunctional and active; (ii) they are more stable with respect tonon-modified sulfamidase; (iii) they are secreted with increasedefficiency compared to non-engineered sulfamidase enzyme; (iv) theintroduction of the ApoB LDLR-BD to produce the finally engineeredsulfamidase did not affect either the functionality or the increasedsecretion efficiency observed in the cells transfected with thepartially engineered sulfamidase. In addition, the finally engineeredconstructs appear to be more stable compared to partially engineeredconstructs.

In Vivo Results in MPS IIIA Mice Injected with Finally EngineeredSulfamidase

We produced AAV2/8 vectors containing one of the finally engineeredsulfamidase (hAATsp-SGSHflag-ApoB) under the liver specific promoterTBG. We obtained very preliminary but extremely encouraging results inMPS-IIIA injected with this viral vector. Adult MPS-IIIA mice weresystemically injected with AAV2/8-TBG- hAATsp-SGSHflag-ApoB. A group ofMPS-IIIA were also injected with AAV2/8-TBG-SGSH (containing the notmodified sulfamidase) as control. The mice were sacrificed one monthafter injection. In the mice injected with the chimeric sulfamidase weobserved higher liver sulfamidase activity and a very strong increase inthe sulfamidase secretion respect to control mice (FIG. 9). Moreover, wedetected a significant increase in SGSH activity into the brain of miceinjected with the chimeric sulfamidase (FIG. 9).

Use of Other Vectors

We completed the production of the AAV2/8 vectors containing all theengineered sulfamidase proteins (partial and final). Specifically,besides the AAV2/8-TBG-hAATsp-SGSHflag-ApoB, we now producedAAV2/8-TBG-hlDSsp-SGSHflag-ApoB; AAV2/8-TBG- hAATsp-SGSHflag andAAV2/8-TBG-hlDSsp-SGSHflag.

These vectors may be used to perform a large in vivo study by thefollowing procedure: MPS-IIIA mice (1 month of age) are injected (by acaudal vein route of administration) with AAV2/8 vectors containing theengineered constructs in order to test the clinical efficacy of thechimeric sulfamidase enzymes. Results are useful to evaluate (i) theefficiency of CNS transduction and (ii) the rescue of CNS pathology inthe treated mice.

BIBLIOGRAPHY

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The invention claimed is:
 1. A method for treating a human subjectdiagnosed with a mucopolysaccharidoses (MPS) pathology, comprisingadministering to the subject a suitable amount of a pharmaceuticalcomposition comprising a viral vector, via a systemic route ofadministration, wherein the MPS pathology is MPS type IIIA, wherein theviral vector comprises a recombinant plasmid suitable for gene therapyof MPS type IIIA, wherein the recombinant plasmid comprises a nucleotidesequence encoding for a chimeric sulfatase, said chimeric sulfataseconsisting essentially of, in the N-terminal to C-terminal sequenceorder of: a) a signal peptide derived from either the humanα-antitrypsin (hAAT) amino acid sequence or the humanIduronate-2-sulfatase (IDS) amino acid sequence; b) a human sulfatasederived amino acid sequence deprived of its signal peptide; c) the ApoBLDLR-binding domain; and wherein the human sulfatase is humansulfamidase.
 2. A method for treating a human subject diagnosed withmucopolysaccharidoses (MPS) pathology comprising administering to thesubject in need thereof a suitable amount of a pharmaceuticalcomposition comprising a chimeric sulfatase; wherein the MPS pathologyis MPS type IIIA, wherein the chimeric sulfatase consists essentially,in the N-terminal to C-terminal sequence order, of: a) a signal peptidederived from either the human α-antitrypsin (hAAT) amino acid sequenceor the human Iduronate-2-sulfatase (IDS) amino acid sequence; b) a humansulfatase derived amino acid sequence deprived of its signal peptide;and c) the ApoB LDLR-binding domain; wherein the human sulfatase ishuman sulfamidase.
 3. The method according to claim 1, wherein thesignal peptide has the sequence of SEQ ID NO: 2 or SEQ ID NO:
 4. 4. Themethod according to claim 1, wherein the encoded human sulfamidasederived amino acid sequence consists essentially of SEQ ID NO:
 8. 5. Themethod according to claim 1, wherein the encoded ApoB LDLR-bindingdomain consists essentially of SEQ ID NO:
 10. 6. The method according toclaim 1, wherein the nucleotide sequence is selected from the groupconsisting of: a) Assembly hAATsp-SGSH-3xflag-ApoB cassette (SEQ ID NO:15), b) Assembly hIDSsp-SGSH-3xflag-ApoB cassette (SEQ ID NO: 17), c)Assembly hAATsp-SGSH-ApoB cassette (SEQ ID NO: 23) and d) AssemblyhIDSsp-SGSH-ApoB cassette (SEQ ID NO: 25).
 7. The method according toclaim 1, wherein the nucleotide sequence is under the control of a liverspecific promoter.
 8. The method according to claim 7 wherein the liverspecific promoter is the human thyroid hormone-globulin (TBG) promoter.9. The method according to claim 1 wherein the viral vector is selectedfrom the group consisting of lentiviral vectors, helper-dependentadenoviral vectors or AAV vectors.
 10. The method according to claim 1wherein the recombinant plasmid is AAV2.1.
 11. The method according toclaim 1 wherein the viral vector is an AAV viral vector.
 12. The methodaccording to claim 11 wherein the viral vector is an AAV serotype8vector.
 13. The method according to claim 2, wherein the signal peptidehas the sequence of SEQ ID NO: 2 or SEQ ID NO:
 4. 14. (Withdrawn/New)The method according to claim 2, wherein the encoded human sulfamidasederived amino acid sequence consists essentially of SEQ ID NO:
 8. 15.The method according to claim 2, wherein the encoded ApoB LDLR-bindingdomain consists essentially of SEQ ID NO:
 10. 16. The method accordingto claim 2, wherein the chimeric sulfatase comprises a sequence selectedfrom the group consisting of: a) hAATsp-SGSH-3xflag-ApoB aminoacidsequence (SEQ ID NO: 16), b) hIDSsp-SGSH-3xflag-ApoB aminoacid sequence(SEQ ID NO: 18), c) hAATsp-SGSH-ApoB aminoacid sequence (SEQ ID NO: 24)and d) hIDSsp-SGSH-ApoB aminoacid sequence (SEQ ID NO: 26).
 17. Themethod according to claim 8 wherein the human thyroid hormone-globulin(TBG) promoter comprises SEQ ID NO:
 27. 18. The method according toclaim 1 wherein the pharmaceutical composition consists of the viralvector.
 19. The method according to claim 2 wherein the pharmaceuticalcomposition consists of the chimeric sulfatase.